This research project involves a team of two cell biologists and a biophysicist who have productively collaborated recently and now are joining together to undertake studies of RNA localization and traffic in the cell nucleus. The proposed research is virtually unique in that intranuclear RNA movements are tracked in living cells, rather than by localization methods that require chemical fixation. The importance of this approach cannot be overstated, since it opens the door to the in vivo investigation of dynamic pathways of intranuclear RNA transport. The two major experimental strategies to be taken, both developed in the P.I?s laboratory, are the fluorescent tagging of endogenous nuclear RNAs and the microinjection of fluorescent RNAs into mammalian cell nuclei. A diffusion-based model for nuclear RNA transport has emerged from this initial work and this will be further investigated. The proposed studies will characterize in detail the intranuclear trafficking patterns of ribosomal RNA as well as beta-globin and c-fos mRNAs. Mutant beta-globin RNAs will be investigated to relate processing defects to intranuclear transport and localization. Studies will also be undertaken of the spliced and polyadenylated but nucleus-restricted RNA, Xist, which is an unusual transcript produced by the inactive X chromosome and is predicted to display very different intranuclear transport properties than mRNAs. Another aspect of the project will address the intranuclear traffic of these RNAs in relation to proteins known to be involved in nuclear import or RNA export, i.e. the Ran-GTPase and TAP.